Cholinergic compositions and uses thereof

ABSTRACT

The invention provides compositions that include conjugates of a cholinergic agent and a fatty acid, preferably cis-docosahexaenoic acid. The conjugates are useful in treating disorders resulting from cerebral ischemia including stroke.

RELATED APPLICATIONS

This application claims priority under 35 U.S.C. §120 from U.S. patentapplication Ser. Nos. 08/651,428, and 08/651,312, U.S. Pat. No.5,795,909, both filed May 22, 1996 the entire disclosures of which areincorporated herein by reference.

BACKGROUND OF THE INVENTION

Stroke is a condition resulting from cerebral ischemia, i.e. a reductionor blockage of blooc flow to the brain, which has neurodegenerativeeffects. About 500,000 Americans suffer strokes each year, 80% of whichare caused by a blood clot blocking one of the cerebral blood vessels.Symptoms of stroke include weakness, numbness or paralysis of the face,arm or leg; sudden loss dimness of vision; loss of speech or difficultyusing or understanding language; sudden, severe unexplained headache; orunexplained dizziness, unsteadiness or sudden falls (articularly ifassociated with one of the above symptoms).

Medications that protect neurons which are at risk following stroke areuseful in reducing neurodegenerative aspects of stroke. Treatments whichcan be administered after a stroke are particularly desirable since itcannot be predicted when onset of stroke will occur. Protection of theneurons from further degeneration permits treatment to restore normalblood flow to the brain (e. using thrombolytics to dissolve blood clotsor surgery to repair a leaking blood vessel) prior to irreversibledebilitating neuronal damage.

Cytidine 5'-diphosphocholine (CDP-choline, citicholine) is an example ofa neuroprotective medication which can exert protective effect whenadministered after a stroke. Cytidine 5'-diphosphocholine is a naturalprecursor of phospholipids such as phosphatidylcholine; when cytidine5'-diphosphocholine is administered, choline and cytidine are releasedinto the systemic circulation. These molecules cross the blood-brainbarrier and are incorporated in membrane phospholipids. CDP-choline hasbeen shown to have a neuroprotective effect in animal models in clinicaltrials, and improves memory and learning deficits in models of aging.Thus CDP-chc appears suitable for treatment of conditions resulting fromcerebral ischemia, such as stroke, and neurodegenerative disordersinvolving loss of cognition, such as Alzheimer's disease.

Fatty acids previously have been conjugated with drugs to help the drugsas conjugates cross the blood brain barrier. For example, DHA(docosahexaenoic acid) is a 22 carbon naturally-occurring, unbranchedfatty acid that previously has been shown to be unusually effective incrossing the blood brain barrier. When DHA is conjugated to a drug, theentire drug-DHA conjugate is transported across the blood-brain barrierand into the brain.

DHA is attached via the acid group to hydrophilic drugs and rendersthese drugs are hydrophobic (lipophilic). DHA is an importantconstituent of the brain and recently has been approved as an additiveto infant formula. It is present in the milk of lactating women. Themechanism of action by which DHA helps drugs conjugated to it cross theblood brain barrier is unknown.

Another example of the conjugation of fatty acids to a drug is theattachment of pipotiazine to stearic acid, palmitic acid, enanthic acid,undecylenic acid or 2,2-dimethyl-palmitic acid. Pipotiazine is a drugthat acts within the central nervous system. The purpose of conjugatingpipotiazine to the fatty acids was to create an oily solution of thedrug as a liquid implant for slow release of the drug when injectedintramuscularly. The release of the drug appeared to depend on theparticular fatty acid selected, and the drug was tested for its activityin the central nervous system.

Lipidic molecules, including the fatty acids, also have been conjugatedwith drugs to render the conjugates more lipophilic than the drug. Ingeneral, increased lipophilicity has been suggests as a mechanism forenhancing intestinal uptake of drugs into the lymphatic system, therebyenhancing the entry of the conjugate into the brain and also therebyavoiding first-pass metabolism of the conjugate in the liver. The typeof lipidic molecules employed have included phospholipic non-naturallyoccurring branched and unbranched fatty acids, and naturally occurringbranched a unbranched fatty acids ranging from as few as 4 carbon atomsto more than 30 carbon atoms. In instance, enhanced receptor bindingactivity was observed (for an adenosine receptor agonist), a waspostulated that the pendant lipid molecule interacted with thephospholipid membrane to acid a distal anchor for the receptor ligand inthe membrane micro environment of the receptor. This increase inpotency, however, was not observed when the same lipid derivatives ofadenosine receptor antagonists were used, and generalizations thus werenot made possible by those studies.

Conjugates containing choline and fatty acid moieties have beensynthesized for various uses. U.S. Pat. No. 5,654,290 describes thepreparation of compounds containing DHA esterified tophosphatidylcholine, lysophosphatidylcholine or a triglyceride. Thecompounds were found useful for delivering DHA into the brain. Yazawa etal described synthesis of polyunsaturated fatty acid-choline esters,including DHA-choline as a function of time iodide (JP 05 43,524).Nishio et al. (Proc. Soc. Exp. Biol. Med. 203:200-208, 1993) found thatcholine-docosahexanoate stimulated phosphatidylcholine-specificphospholipase C activity. Another reference (JP 62 45,536) disclosed avariety of fatty acid-choline esters for enhancing oral, nasal andvaginal absorption of pharmaceuticals. U.S. Pat. No. 5,466,841 describesphospholipids containing choline and two different unsaturated fattyacids (on of which can be DHA). None of the foregoing compoundscontaining choline conjugated to one or more fatty acid moieties havebeen used in the treatment of stroke or cognitive disorders.

SUMMARY OF THE INVENTION

It has now been discovered that a covalent conjugates of a fatty acidand a cholinergic agent is useful in the treatment of stroke.Unexpectedly, DHA-cholinergic agent conjugates reduce the effects ofcerebral ischemia in an animal model of stroke, even when administeredseveral hours after the ischemic event. Furthermore, DHA-cholinergicagent conjugates unexpectedly protect cortical neurons selectivelyfollowing cerebral ischemia. The conjugates are believed useful forthrombotic, embolic, and hemorrhagic stroke.

The cholinergic agent preferably is conjugated directly to the fattyacid via the COOH of the fatty acid, without a linker.

According to one aspect of the invention, a pharmaceutical compositionis provided. A composition contains a covalent conjugate of acholinergic agent and a fatty acid having 12-26 carbons, in an amounteffective to treat stroke, and a pharmaceutically acceptable carrier.Cholinergic agent as used herein is a cholinergic agonist. These areagents that stimulate nerve systems that are activated by acetylcholine.Examples of cholinergic agents are: aceclidine; demecarium bromide;dexpanthenol; echothiophate iodide; isoflurophate; neostigmine bromide;neostigmine methylsulfate; physostigmine; physostigmine salicylate;physostigmine sulfate; pyridostigmine bromide, acetylcholine chloride,arecoline HBr, bethanechol chloride, carbachol, cis-dioxolane,(+)-,(4-hydroxy-2-butynyl)-1-trimethylammonium m-chlorocarbonilate chloride,methacholine chloride, metoclopramide HCI, muscarine chloride,(±)-,muscarine chloride(+)-, nicotine tartrate,S(-)-,cis-2-methyl-5-trimethylammoniummethyl-1,3-oxathiolane iodide (OXA-22),oxotremorine, oxotremorine sesquifumarate or pilocarpine HCl,(+)-. Incertain embodiments, the cholinergic agent is not acetylcholinechloride, arecoline HBr, bethanechol chloride, carbachol,cis-dioxolane,(+)-, (4-hydroxy-2-butynyl)-1-trimethylammoniumm-chlorocarbonilate chloride, methacholine chloride, metoclopramide HCl,muscarine chloride,(±)-, muscarine chloride(+)-, nicotinetartrate,S(-)-, cis-2-methyl-5-trimethylammoniummethyl-1,3-oxathiolaneiodide OXA-22 oxotremorine, oxotremorine sesquifumarate or pilocarpineHCl,(+)-.

Preferably, the fatty acid is an unbranched, naturally occurring fattyacid. More preferably, the fatty acid has 16-22 carbons. It also ispreferred that the fatty acid and choline are conjugated via an esterbond between the COOH of the fatty acid and the OH of the choline.Unbraiiched common naturally occurring fatty acids include C12:0 (lauricacid), C14:0 (myristic acid), C16:0 palmitic acid), C16:1 (palmitoleicacid), C16:2, C18:0 (stearic acid), C18:1 (oleic acid), C18:1-7(vaccenic), C18:2-6 (linoleic acid), C18:3-3 (α-linolenic acid), C18:3-5(eleostearic), C1 8:3-6 (β-linolenic acid), C18:4-3, C20:1 (gondoicacid), C20:2-6, C20:3-6 (dihomo-y-linolenic acid), C20:4-3, C20:4-6(arachidonic acid), C20:5-3 (eicosapentaenoic acid), C22:1 (docosenoicacid), C22:4-1 (docosatetraenoic acid), C22:5-6 (docosapentaenoic acid),C22:5-3 (docosapentaenoic), C22:6-3 (docosahexaenoic acid) and C24:1-9(nervonic). Highly preferred unbranched, naturally occurring fatty acidsare those with between 16 and 22 carbon atoms. The most preferred fattyacid is docosahexaenoic acid.

Most preferably, the composition is ##STR1##

The pharmaceutical composition further can comprise an anti-stroke agentother than the covalent conjugate. In certain embodiments, theanti-stroke agent is selected from the group consisting of antiplateletagents, anticoagulation agents, cholinergic agents, thrombolytic agentsincluding plasminogen activators, antithrombotics, neuroprotectiveagents, platelet activating factor antagonists, platelet aggregationinhibitors, post-stroke and post-head trauma treatments, cerebralischemia agents, basic fibroblast growth factors and steroids. Mostpreferably, the anti-stroke agent is selected from the group consistingof citicholine, dizocilpine, urokinase tissue plasminogen activation andlexipafant.

According to another aspect of the invention, a kit is provided. The kitis a package which houses a container which contains the covalentconjugate of the invention and also houses instructions foradministering the covalent conjugate to a stroke victim.

According to another aspect of the invention, a second kit is provided.This kit includes a package which houses a first container whichcontains the covalent conjugate of the invention and also houses asecond container containing an anti-stroke agent other than the covalentconjugate.

In the kits of the invention, the preferred fatty acids, bonds,cholinergic agents, convalent conjugate and anti-stroke agent other thanthe covalent conjugate are as described above.

According to another aspect of the invention, a method is provided fortreating stroke. The method involves administering to a subject in needof such treatment a covalent conjugate of a cholinergic agent and afatty acid having 12-26 carbons in an amount effective to treat stroke.The preferred fatty acids, bonds, cholinergic agents and covalentconjugates are as described above. I method also can involveco-administering to the subject an anti-stroke agent other than thecovalent conjugate. Preferred anti-stroke agents are as described above.

According to another aspect of the invention, a method is provided forprotecting cortical cells from ischemia-induced cell death. The methodinvolves contacting the cortical cells which have been exposed toischemic conditions sufficient to induce cell death with a covalentconjugate of a cholinergic agent and a fatty acid having 12-26 carbonsin an amount effective to protect the cortical cells against cell deathwhich would otherwise result from the ischemic conditions. Preferredfatty acids, bonds, cholinergic agents and covalent conjugates are asdescribed above.

According to another aspect of the invention, a method is provided forselectively protecting cortical cells of a subject from stroke-inducedcell death. The method involves administering to subject in need of suchtreatment a covalent conjugate of a cholinergic agent and a fatty acidhaving 12-26 carbons in an amount effective to protect the corticalcells from stroke-induced cell death. Preferred fatty acids, bonds andcovalent conjugate are as described above. These and other aspects theinvention are described in greater detail below.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the dose response data for the locomotor activity of miceinjected with different doses of DHA-choline.

Fig. 2 shows the dose response data for the locometer activity of mimeinjected with different doses of DHA-clorine as a function of dose.

DETAILED DESCRIPTION OF THE INVENTION

Choline is a naturally occurring alcohol which is a component of lipids(e.g. phosphatidylcholine) and the neurotransmitter acetylcholine.Choline has the following structure: ##STR2##

cis-docosahexaenoic acid (DHA) is a naturally occurring fatty acid. Itis an unbranched chain fatty acid with six double bonds, all cis. Itsstructure is as follows: ##STR3##

DHA can be isolated, for example, from fish oil or can be chemicallysynthesized. These methods, however, can generate trans isomers, whichare difficult and expensive to separate and which may present safetyproblems in humans. The preferred method of production is biologicalsynthesis to produce the all cis isomer. The preferred source of DHA isfrom Martek Biosciences Corporation of Columbia, Md. Martek has apatented system for manufacturing DHA using microalgae which synthesizeonly a single isomer of DHA, the all cis isomer. Martek's patentsinclude U.S. Pat. Nos. 5,374,657, 5,492,938, 5,407,957 and 5,397,591.

DHA also is present in the milk of lactating women, and Martek'slicensee has obtained approval in Europe of DHA as a nutritionalsupplement for infant formula.

It is known that DHA can be unstable in the presence of oxygen. Tostabilize DHA and its conjugates it is important to add anti-oxidants tothe material after it is synthesized. One method of stabilization is tomake-up the newly synthesized material in the following solution: 100 gneat DHA-choline plus 100 g of vehicle (100 ml propylene glycol, 70 mgalpha-tocopherol, 5 mg dialaurylthiodipropionic acid, 50 mg ascorbicacid) prepared and held under argon in amber, sealed vials and stored atfour degrees centigrade. The following anti-oxidants may also beemployed: ascorbic acid, ascorbyl palmitate, dilauryl ascorbate,hydroquinone, butyated hydroxyanisole, sodium meta bisulfite, t-βcarotene and α-tocopherol. A heavy metal chelator such asethylenediarnine tetra-acetic acid (EDTA) may also be used.

In one aspect of the invention, the conjugate is prepared as aquaternary ammonium salt. The anion preferably is selected from thegroup consisting of I⁻, Cl⁻, OH⁻, F⁻ and Br⁻. Most preferably the anionis I⁻.

In another aspect of the invention, cocktails of the choline-fatty acidconjugate and another anti-stroke agent can be prepared foradministeration to subjects having a need for such treatment. One ofordinary skill in the art is familiar with a variety of antistrokeagents which are used in the medical arts to treat stroke (thrombotic,embolic and/or hemorrhagic stroke). Such agents include antiplateletagents, anticoagulation agents, thrombolytic agents includingplasminogen activators, antithrombotics, neuroprotective agents,platelet activating factor antagonists, platelet aggregation inhibitors,post-stroke and post-head trauma treatments, cerebral ischemia agents,basic fibroblast growth factors and steroids

Antiplatelet agents, which inhibit platelet aggregation, includeaspirin, ticlopidine and dipyridamole.

Anticoagulation agents reduce or prevent the coagulation of bloodcomponents and thus reduce or prevent clot formation; commonanticoagulation agents include coumarin and heparin.

Thrombolytic agents function by lysing the clot which causes thethromboembolic stroke. Commonly used thrombolytic agents includeurokinase, streptokinase and tissue plasminogen activator (alteplase,tPA). Various modified forms of tPA ("modified tPA") have beencharacterized and are known to those skilled in the art. Modified tPAincludes, but is not limited to, variants having deleted or substitutedamino acids or domains, variants conjugated to other molecules, anvariants having modified glycosylation. For example, PCT Publication No.W093/24635 discloses tPA variants having an extra glycosylation site atany of the amino acid positions 103-105 and the native glycosylationsite removed at position 117 of the native human tPA. The amino acidnumber refers to the amino acid in that position of the mature,wild-type tPA polypeptide as disclosed in U.S. Pat. No. 4,766,075. Thedisclosed variants may also include at least one amino acid substitutedin the 296-299 position with alanine and/or a substitution of the aminoacids at positions 274-277 of wild type tPA (phenylalanine, arginine,isoleucine, lysine) with leucine, histidine, serine, and threonine,respectively. Triple mutants of tPA also are disclosed, including thespecific molecule: T103N, N117Q, KHRR (296-299) AAAA t-PA (TNK t-PA). EP352,119 discloses vampire bat tPAs (Bat-PAs (H), (I), and (L)). Vampirebat-PAs are variants of native tPA having a variety of sequencemodifications. Suzuki et al., (J. Cardiovasc. Pharmacal.22:834-840,1993) disclose tPA variants in which a cysteine at position 84 of thegrowth factor domain of native tPA is replaced by serine (C84S tPA).Although this variant retains the functional activity of native tPA, ithas been shown to have a longer in vivo half life than native tPA.

Variants of tPA have been developed which retain tPA functionality buthave reduced clearance rates. These variants include tPA molecules withdeleted amino acids or domains, such as those described by Johannessenet al. (Throm. Haemostas. 63:54-59, 1990) and Sobel et al. (Circulation81:1362-73, 1990); tPA molecules which have amino acid substitutions inthe regions of 63-72 and 42-49, such as those described by Ahem et al.(J Biol. Chem. 265:5540, 1990); and tPA molecules which have a glutamicacid substituted for the arginine at position 275 of the native tPAmolecule such as that described by Hotchkiss et al. (Throm. Haemostas.55:491, 1987). tPA molecules conjugated to other molecules have alsobeen found to have decreased clearance rates. For example, conjugationof tPA to polyethylene glycol has been shown to reduce the clearancerate of tPA, as disclosed in EP-A304,311. Conjugation of a tPA moleculeto a monoclonal antibody has been shown to increase the half-life of tPAin vivo (EP A339,505).

Modification of glycosylation on native tPA has also been found to havean effect on clearance rates of tPA. PCT application W089/11531discloses several tPA variants having additional glycosylation sites,which also have decreased clearance rates. Other research has describedtPA variants with reduced glycosylation, which also exhibit decreasedclearance rates Martin et al., Fibrinolysis 4:9, 1990). Each of theabove references is hereby incorporated by reference.

Antithrombotics include anagrelide hydrochloride; bivalirudin ;dalteparin sodium; danaparoid sodium; dazoxiben hydrochloride; efegatransulfate; enoxaparin sodium; ifetroban; ifetroban sodium; tinzaparinsodium; and trifenagrel.

Neuroprotective agents include dizocilpine maleate.

Platelet activating factor antagonists include lexipafant.

Platelet aggregation inhibitors include acadesine; beraprost; beraprostsodium; ciprostene calcium; itazigrel; lifarizine; oxagrelate.

Post-stroke and post-head trauma agents include citicoline sodium.

Cerebral ischemia agents include dextrorphan hydrochloride.

The conjugates of the invention, when used alone or in cocktails, areadministered in effective amounts. In general, an effective amount willbe that amount necessary to inhibit stroke or the neurodegenerativeeffects thereof. An effective amount is one sufficient to reduce in vivobrain injury resulting from the stroke. A reduction of brain injury isany prevention of injury to the brain which otherwise would haveoccurred in a subject experiencing a stroke absent the treatment of theinvention. Several physiological parameters may be used to assessreduction of brain injury, including smaller infarct size, improvedregional cerebral blood flow, and decreased intracranial pressure, forexample, as compared to pretreatment patient parameters, untreatedstroke patients or, in the case of treatment with cocktails, strokepatients treated with antistroke agents alone (i.e. without theconjugate of the invention). These parameters can be monitored usingstandard diagnostic procedures including magnetic resonance imaging(MRI), computed tomographic (CT) scans, cerebral angiography,noninvasive carotid evaluations by ophthalmodynamometry,oculoplethysmography, range-gated pulsed-Doppler assessment andtranscranial Doppler assessment, and the like. When administered to asubject, effective amounts will depend, of course, on the particularcondition being treated; the severity of the condition; individualpatient parameters including age, physical condition, size and weight;concurrent treatment; frequency of treatment; and the mode ofadministration. These factors are well known to those of ordinary skillin the art and can be addressed with no more than routineexperimentation. It is preferred generally that a maximum dose be used,that is, the highest safe dose according to sound medical judgment.

Dosage may be adjusted appropriately to achieve desired drug levels,locally or systemically. generally, daily oral doses of active compoundswill be from about 0.01 mg/kg per day to 1000 g/kg per day. It isexpected that i.v. doses in the same range will be effective. In theevent that the response in a subject is insufficient at such doses, evenhigher doses (or effective higher doses by a different, more localizeddelivery route) may be employed to the extent that patient tolerancepermits. Continuous IV dosing over, for example 24 hours or multipledoses per day are contemplated to achieve appropriate systemic levels ofcompounds.

When administered, the formulations of the invention are applied inpharmaceutically acceptable compositions. Such preparations mayroutinely contain salts, buffering agents, preservatives, compatiblecarriers, and optionally other therapeutic ingredients. When used inmedicine the salts should be pharmaceutically acceptable, butnon-pharmaceutically acceptable salts may conveniently be used toprepare pharmaceutically acceptable salts thereof and are not excludedfrom the scope of the invention. Such pharmacologically andpharmaceutically acceptable salts include, but are not limited to, thoseprepared from the following acids: hydrochloric, hydrobromic, sulphuric,nitric, phosphoric, maleic, acetic, salicylic, p-toluene sulfonic,tartaric, citric, methane sulfonic, formic, malonic, succinic,naphthalene-2-sulfonic, and benzene sulfonic. Also, pharmaceuticallyacceptable salts can be prepared as alkaline metal or alkaline earthsalts, such as sodium, potassium or calcium salts.

Suitable buffering agents include: acetic acid and a salt (1-2% W/JV);citric acid and a salt (1-3% W/V); and phosphoric acid and a salt(0.8-2% W/V).

Suitable preservatives include benzalkonium chloride (0.003-0.03% W/V);chlorobutanol (0.3-0.9% W/V); parabens (0.01-0.25% W/V) and thimerosal(0.004-0.02% W/V).

The active compounds of the present invention may be a pharmaceuticalcomposition having a therapeutically effective amount of a conjugate ofthe invention optionally included in a pharmaceutically-acceptablecarrier. The term "pharmaceutically-acceptable carrier" as used hereinmeans one or more compatible solid or liquid filler, dilutants orencapsulating substances which are suitable for administration to ahuman or other animal. The term "carrier" denotes an organic orinorganic ingredient, natural or synthetic, with which the activeingredient is combined to facilitate the application. The components ofthe pharmaceutical compositions are capable of being commingled with themolecules of the present invention, and with each other, in a mannersuch that there is no interaction which would substantially impair thedesired pharmaceutical efficacy.

Compositions suitable for parenteral administration convenientlycomprise a sterile preparation of the conjugates of the invention. Thispreparation may be formulated according to known methods.

The sterile preparation thus may be a sterile solution or suspension ina non-toxic parenterally-acceptable diluent or solvent. In addition,sterile, fixed oils are conventionally employed as a solvent orsuspending medium. For this purpose any bland fixed oil may be employedincluding synthetic mono or di-glycerides. In addition, fatty acids suchas oleic acid find use in the preparation of injectables. Carrierformulations suitable for oral, subcutaneous, intravenous,intramuscular, etc. can be found in Remington's Pharmaceutical Sciences,Mack Publishing Company, Easton, Pa.

The invention is used in connection with treating subjects having orsuspected of having a stroke. A subject as used herein means humans,primates, horses, cows, pigs, sheep, goats, dogs, cats and rodents.

A variety of administration routes are available. The particular modeselected will depend of course, upon the particular drug selected, theseverity of the disease state being treated and the dosage required fortherapeutic efficacy. The methods of this invention, generally speaking,may be practiced using any mode of administration that is medicallyacceptable, meaning any mode that produces effective levels of theactive compounds without causing clinically unacceptable adverseeffects. Such modes of administration include oral, rectal, sublingual,topical, nasal, transdermal or parenteral routes. The term "parenteral"includes subcutaneous, intravenous, intramuscular, or infusion.Intravenous routes are preferred.

The compositions may conveniently be presented in unit dosage form andmay be prepared by any of the methods well known in the art of pharmacy.All methods include the step of bringing the conjugates of the inventioninto association with a carrier which constitutes one or more accessoryingredients. In general, the compositions are prepared by uniformly andintimately bringing the compounds into association with a liquidcarrier, a finely divided solid carrier, or both, and then, ifnecessary, shaping the product.

Compositions suitable for oral administration may be presented asdiscrete units such as capsules, cachets, tablets, or lozenges, eachcontaining a predetermined amount of the active compound. Othercompositions include suspensions in aqueous liquors or non-aqueousliquids such as a syrup, an elixir, or an emulsion.

Other delivery systems can include time-release, delayed release orsustained release delivery systems. Such systems can avoid repeatedadministrations of the active compounds of the invention, increasingconvenience to the subject and the physician. Many types of releasedelivery systems are available and known to those of ordinary skill inthe art. They include polymer based systems such as polylactic andpolyglycolic acid, polyanhydrides and polycaprolactone; nonpolymersystems that are lipids including sterols such as cholesterol,cholesterol esters and fatty acids or neutral fats such as mono-, di andtriglycerides; hydrogel release systems; silastic systems; peptide basedsystems; wax coatings, compressed tablets using conventional binders andexcipients, partially fused implants and the like. In addition, apump-based hardware delivery system can be used, some of which areadapted for implantation.

A long-term sustained release implant also may be used. "Long-term"release, as used herein, means that the implant is constructed andarranged to deliver therapeutic levels of the active ingredient for atleast 30 days, and preferably 60 days. Long-term sustained releaseimplants are well known to those of ordinary skill in the art andinclude some of the release systems described above.

EXAMPLES

Synthesis of DHA-Choline

(A) Synthesis of 2-dimethylaminoethyl docosahexaenoate: ##STR4##

To a solution of docosahexaenoic acid (0.986 g, 3.0 mmol) in CH₃ CN (6.0mL) was added carbonyldiimidazole (0.535 g, 3.3 mmol) in one portion at0° C. The mixture was allowed to warm to room temperature and stirred atroom temperature for 30 min. TLC showed a complete reaction (1:1EtOAc/hexane). N,N-dimethylaminoethanol (0.89 g, 10.0 mmol) was addeddropwise followed by addition of 4-dimethylaminopyridine (0.073 g, 0.60mmol) The mixture was stirred at room temperature overnight. The solventwas removed by rotary evaporation and the residue was purified on silicagel using 72% EtOAc/hexane, 80% EtOAC/hexane and 100% EtOAc with 0.5%MeOH each to provide the product, 2-dimethylaminoethyl docosahexaenoate(1.098 g, 92%), as a light yellow oil. The product was stored at -20° C.with small amount of β-carotene.

(B) Analysis of the product:

(1) TLC:

Rf (100% EtOAc)

DHA choline precursor 0.19

DHA 0.55

N,N-dimethylethanolamine 0.05

Rf (butanol:pyridine:H₂ O 85:10:5)

DHA choline precursor 0.32

DHA 0.81

N,N-dimethylethanolamine 0.16

(2) Mass spectrum: M+400

(3) Elemental analysis: calculated for C₂₆ H₄,NO₂ : C% 78.15, H% 8.62,N% 2.92. Found: C% 78.11, H% 10.54, N% 3.42.

(4) NMR:

¹ H NMR (CDC1₃) δ5.44-5.22 (m, 12 H), 4.13 (t, J=5.75 Hz, 2H), 2.86-2.70(m, 10 H), 2.52 (t,J=5.75 Hz, 2 H), 2.40-2.30 (m, 4 H), 2.24 (s, 6 H),2.03 (pent, J=7.50 Hz, 2 H), 0.98 (t, J=7.50 Hz, 3 H).

¹³ C NMR (CDC1₃) δ172.65, 131.69, 128.95, 128.25, 127.93, 127.79,127.64, 127.58, 126.74, 61.84, 57.54, 45.41, 33.83, 25.35, 25.32, 25.26,22.49, 20.28, 14.01.

(5) Solubility: soluble in EtOAc, Et₂ O, CH₂ C₂, CHCl₃, EtOH insolublein H₂ O

(6) Stability: turns dark when exposed in the air for several days,should be kept at -20° C. under Argon.

(C) Synthesis of Docosahexaenoyl Choline Iodide ##STR5##

Molecular Formula: C₂₇ H₄₄ NO_(2I;) MW: 541.56.

To a solution of 2-dimethylaminoethyl docosahexaenoate (1.12 g, 2.80mmol) in CH₂ Cl₂ (10.0 mL) was added iodomethane (0.80 g, 5.60 mmol)dropwise at room temperature. The mixture was stirred at roomtemperature for 3 hr. The solvent and excess reagent was removed underreduced pressure and the residue was triturated with hexanes. Themixture was centrifuged and the supernatant was removed. The residue wasdried under reduced pressure to provide the product, docosahexaenoylcholine iodide (1.46 g, 96%), as an off white solid.

(D) Analysis of the product:

(1) Mass spectrum: M⁺ --I414

(2) NMR

¹ H NMR (CDCl₃) δ5.46-5.21 (m, 12 H), 4.55 (br s, 2 H), 4.11-4.02 (m, 2H), 3.51 (s, 9 H), 2.862.74 (m, 10 H), 2.48-2.30 (m, 4 H), 2.04 (pent,J=7.50 Hz, 2 H), 1.03 (t, J=7.50 Hz, 3 H).

¹³ C NMR (CDCl₃) δ171.81, 131.78, 129.51, 128.32, 128.20, 128.05,127.77, 127.59, 127.16, 126.72, 64.96, 57.56, 54.55, 33.72, 25.37,25.27, 22.15, 20.30, 14.03.

(3) Solubility: soluble in CH₂ Cl₂, CHCl₃, EtOAc, EtOH insoluble inhexanes, Et₂ O.

5.5 mg dissolved in 1.0 mL ascorbic acid in saline with 0.02 mLdetergent

7.8 mg dissolved in 1.0 mL 10% albumin in saline with 0.01 mL detergent

(4) Stability: ¹ H NMR analysis of the compound which was exposed in theair for 5 days showed that the compound had not decomposed; however, ifit is exposed in the air for too long will decompose. The compoundshould be kept at -20° C. under Argon. Also purification of the compoundon neutral alumina will result in other peaks on ^(1H) NMR.

Activity of DHA-Choline

(A) Locomotor activity studies:

A dose-response study of DHA-choline-induced locomotor depression wasconducted using 40 Digiscan locomotor activity testing chambers(40.5×40.5×30.5 cm) housed in sets of two, within sound attenuatingchambers. A panel of infrared beams (16 beams) and corresponding photodetectors were located in the horizontal direction along the sides ofeach activity chamber. A 7.5W incandescent light above each chamberprovided dim illumination. Fans provided an 80-dB ambient noise levelwithin the chamber. Separate groups of 8 non-habituated maleSwiss-Webster mice (Hsd:ND4, aged 2-3 mo.) were injected via theintraperitoneal route (i.p.) with either vehicle or DHA-choline (0.5,2.5, 5, 10, 20 or 40 mg/kg), 20 minutes prior to locomotor activitytesting. In all studies, the total distance (cm traversed in thehorizontal plane) was measured for 2 hours within 10 min periods.

The figures show the dose response data for the locomotor activity(Stewart et al., Psychopharmacol 60:281, 1979) of mice injected withdifferent doses of the compound. The figures show average distance per10 min as a function of time (FIG. 1) and dose (FIG. 2) 20 minutesfollowing DHA-choline pretreatment. The period 0-30 min was selected foranalysis of dose-response data because this is the time period in whichDHA-choline produced maximal effects. The mean average distance per 10min for this 30 min period were fit to a linear function of log₁₀ doseof the descending portion of the dose-effect curve (0.5 to 40 mg/kg doserange). The ID₅₀ dose producing 1/2 maximal depressant activity (wheremaximal depressant activity =0 cm/30 min) was calculated as 12.9 mg/kg.

A one-way analysis of variance conducted on total distance/10 min forthe 0-30 time period indicated a significant overall effectF(6,49)=10.5, p<0.001; planned comparisons (a priori contrast) againstthe vehicle control showed a significant difference for 2.5, 10, 20 and40 mg/kg (all ps<05 denoted on FIG. 1 with an asterisk).

Thus, these data demonstrate that DHA-choline inhibits locomotoractivity in a dose-dependent manner.

(B) Evaluation of DHA-choline as an anti-stroke compound

Two sets of experiments were carried out. In the first set, rats wereadministered with 50 mg/kg of DHA-choline i.p. at 30 minutes prior toocclusion of the middle cerebral artery of the left side of the brainusing a standard highly reproducible animal model of stroke (Karpiak etal., J. Neurosci Res.30:512-520, 1991). Occlusion of the middle cerebralartery inhibits blood flow to a major portion of the left cortical andsubcortical regions of the brain. After a two hour period, the occludedblood vessel was opened to allow reperfusion of the brain, andanesthesia was terminated. Each animal received additional doses of 50mg/kg of DHA-choline 24 and 48 hours later.

At the end of the three days, the animals were tested for neurologicaldeficits and evaluated by the standard test scores of 0 to 5 (0= normal,no deficit; 1= extend forepaw on contralateral side; 2= circling animal;3= loss of righting reflex; 4= animal cannot stand; 5= dead). All of thevehicle treated rats showed a typical disability to extend thecontralateral front paw, and circled on the side of the affected legwhile walking. These deficits are primarily associated with corticaldamage. All of the drug treated animals did not show paw extensiondisability and walked normally in a straight line.

The animals were sacrificed, perfused with formalin fixative, brainswere sliced into seven 2 mm thick coronal sections, and stained withtriphenyltetrazolium chloride (Watson et al., J. Neurosci. Methods53:203-208, 1994). In this test, tissue that contains intactmitochondria stains red, whereas dead tissue with damaged mitochondriapicks up no stain and remains white. Each unstained area on the leftside of a section was measured and compared to the total area of thecontrol non-occluded right side of the same section of the brain. Thearea of damaged (white) brain tissue cells was calculated as a percentof the intact right side of a section. Table 1 demonstrates that thenumber of dead cells decreased by about 50% for the animals treated withDHA-choline. Therefore, DHA-choline rescued brain tissue from effects ofocclusion when it is administered at 30 minutes prior to the initiationof ischemia. (n=5 per group.)

In the second set of experiments (n=5), the identicalocclusion-reperfusion rat model was used; only the time ofadministration of the drug was changed. DHA-choline was injected i.p. ata dose of 50 mg/kg at one hour after the beginning of the reperfusion,i.e., at three hours after the initiation of the stroke event. Controls(n=5) received an injection of the vehicle instead of the drug accordingto the same protocol. Surprisingly, the drug-treated brains showedapproximately 50% the infarct volume, i.e., identical to the resultsobserved in the animals that had received a pretreatment.

                  TABLE I                                                         ______________________________________                                        Effect of NMI 96103 on MCA occlusions of rat brain                              Infarct volume (% of control non-occluded side of brain)                                                       DHA-choline at 3                               DHA-choline at 30 min prior to hours after                                  Group No. Vehicle occlusion occlusion                                       ______________________________________                                        1      33      22                15                                             2 37 19 17                                                                    3 37 16 25                                                                    4 35 20 21                                                                    5   20                                                                        Average 35.5 19.3 19.6                                                      ______________________________________                                    

Unexpectedly, there was a complete rescue of the cortical cells inanimals that received DHA-choline, regardless of whether DHA-choline wasadministered prior to the onset of the or at three hours after the onsetof occlusion. All animals treated with DHA-choline had only subcorticalinfarcts with no incidence of cortical infarcts. These results suggestthat DHA-choline is a neuroprotective drug which will be effective fortreatment of stroke and which, unexpectedly, completely rescues corticalneurons from death following cerebral ischemia.

Other aspects of the invention will be clear to the skilled artisan andneed not be repeated here. All patents, published patent applicationsand literature cited herein are incorporated by reference in theirentirety.

While the invention has been described with respect to certainembodiments, it should be appreciated that many modifications andchanges may be made by those of ordinary skill in the art withoutdeparting from the spirit of the invention. It is intended that suchmodification, changes and equivalents fall within the scope of thefollowing claims.

We claim:
 1. A method for treating stroke comprisingadministering to asubject in need of such treatment an amount of a covalent conjugate of acholinergic agent and a fatty acid having 12-26 carbons in an amounteffective to treat stroke.
 2. The method of claim 1, wherein the fattyacid is docosahexaenoic acid.
 3. The method of claim 1 with the provisothat the cholinergic agent is not acetylcholine chloride, arecoline HBr,bethanechol chloride, carbachol, cis-dioxolane,(+)-,(4-hydroxy-2-butynyl)-1-trimethylammonium m-chlorocarbonilate chloride,methacholine chloride, metoclopramide HCl, muscarine chloride,(+)-,muscarine chloride(+)-, nicotine tartrate,S(-)-,cis-2-methyl-5-trimethylammoniummethyl-1,3-oxathiolane iodide (OXA-22),oxotremorine, oxotremorine sesquifumarate or pilocarpine HCl,(+)-. 4.The method of claim 3, further comprising administering to the subjectan anti-stroke agent. other than the covalent conjugate and wherein theanti-stroke agent is selected from the group consisting of antiplateletagents, anticoagulation agents, cholinergic agents, thrombolytic agentsincluding plasminogen activators, antithrombotics, neuroprotectiveagents, platelet activating factor antagonists, platelet aggregationinhibitors, post-stroke and post-head trauma treatments, cerebralischemia agents, basic fibroblast growth factors and steroids.
 5. Themethod of claim 3 wherein the anti-stroke agent is selected from thegroup consisting of citicholine, dizocilpine, alteplase, urokinase, andlexipafant.
 6. A method for protecting cortical cells fromischemia-induced cell death comprisingcontacting the cortical cellswhich have been exposed to ischemic conditions sufficient to induce celldeath with a covalent conjugate of a cholinergic agent and a fatty acidhaving 12-26 carbons in an amount effective to protect the corticalcells against cell death which would otherwise result from the ischemicconditions.
 7. The method of claim 6, wherein the fatty acid isdocosahexaenoic acid.
 8. A method for selectively protecting corticalcells of a subject from stroke-induced cell deathcomprisingadministering to a subject in need of such treatment acovalent conjugate of a cholinergic agent and a fatty acid having 12-26carbons in an amount effective to protect the cortical cells from strokeinduced cell death.
 9. The method of claim 8, wherein the fatty acid isdocosahexaenoic acid.
 10. The method of claim 1, wherein the fatty acidis an unbranched, naturally-occurring fatty acid.
 11. The method ofclaim 10, wherein the fatty acid has 14-22 carbons.
 12. The method ofclaim 1, wherein the fatty acid is conjugated to the cholinergic agentvia an ester bond involving the COOH of the fatty acid.
 13. The methodof claim 6, wherein the fatty acid is an unbranched, naturally-occurringfatty acid.
 14. The method of claim 13, wherein the fatty acid has 14-22carbons.
 15. The method of claim 6, wherein the fatty acid is conjugatedto the cholinergic agent via an ester bond involving the COOH of thefatty acid.
 16. The method of claim 8, wherein the fatty acid is anunbranched, naturally-occurring fatty acid.
 17. The method of claim 9,wherein the fatty acid has 14-22 carbons.
 18. The method of claim 8,wherein the fatty acid is conjugated to the cholinergic agent via anester bond involving the COOH of the fatty acid. a second containercontaining an anti-stroke agent other that the covalent conjugate.